PROJECT SUMMARY/ABSTRACT Treatment for end-stage organ failure is restricted by the critical shortage of donor organs with the organ waiting list currently at 100,000 requests and it is increasing by 5% every year. The problem is not different for liver, which this study focuses on - about 4,000 people die in the US due to lack of a transplantable organ, and the lack of donor organs is considered a major health crisis. In addition, since transplantation can often be the solution to many aging related diseases, the hidden demand is estimated to be far beyond the current levels. This situation has been a major driving force behind the rise of tissue engineering, with the market for organ failure treatments estimated at about $80 billion. However, over two decades of work aimed at building tissues from the ground up has not succeeded in creating large-scale tissues that can be clinically implanted to address the void in organ replacement therapies. Further, despite intense efforts on the stem cell front, including those from our group, the lack of a reliable cell source for primary adult hepatocytes for use in cell therapies persists and is unlikely to be resolved in the near future. Interestingly enough, there are many potential organ donors that are not considered for transplantation because the organs are damaged. For example, accident victims who arrive at the hospital after cardiac death are not eligible donors because of excessive ischemic damage; even a slight ischemic damage (>30min warm ischemia or >16hrs cold ischemia) is known to lead to complications in the long term with significantly reduced graft survival at 6 months. By some estimates, potential number of Donors after Cardiac Death (DCD) is over 200,000 per year in the US, and about 6,000 are considered to be only marginally damaged. Our long-term goal is to engineer transplantable liver grafts for curing or treating relevant liver diseases. The objective of the proposed study is to develop humanized reengineered liver grafts as a viable in vitro liver model. During the mentored phase (K99) of the award, two essential tools will be developed to reach this goal: 1) a liver perfusion system, which will enable recovery of healthy hepatocytes from marginal donor livers. This technology is expected to lead to establishment of a currently untapped source of adult human hepatocytes that will fill the need for human cells until stem cell approaches mature and become safe and efficient for clinical use. 2) a whole organ perfusion- decellularization and recellularization methodology. The objective here is to develop a novel scaffold for tissue engineering, which supports cell attachment and function and is vascularizable. During the independent phase (R00) of the award, the primary goal is the scaling of the methods developed in K99 phase to large animal models and ultimately human organs. The work proposed in this project is expected to i) establish marginal livers as a reliable source of primary hepatocytes, ii) establish decellularized liver slices as novel 3D cell culture platform to study the role of ECM, iii) develop humanized rat liver grafts as a three dimensional liver model for pharmaceutical studies, and iv) lead to the development of reengineered liver grafts to treat liver diseases. While this work utilizes liver as the model organ, the results of this work will also have a positive impact by establishing the basis of future sophisticated organ engineering techniques that incorporate multiple different cell types and can be translated to other organs (such as pancreas to create vascularized patches for pancreatic -cell transplantation), and may ultimately lead to development of entire organs in vitro.